JP6873995B2 - Hepcidin detection kit - Google Patents

Description

Cross-reference This application is incorporated by reference in its entirety, US Patent Provisional Application Nos. 62 / 260,106 filed on November 25, 2015 and US Patent Provisional Application No. 62, filed on December 4, 2015. Claim the benefits of No. 62 / 263,245.

Sequence Listing This application contains a sequence listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was made on November 18, 2016, named 44546-710_601_SL.txt, and is 4,024 bytes in size.

Background of the Invention Iron is an essential trace element required for the growth and development of living organisms. In mammals, iron content is regulated by controlling iron absorption, iron reuse, and iron release from cells that store iron. Iron is mainly absorbed by enterocytes in the duodenum and upper jejunum. Iron is reused from reticular endothelial macrophages in the bone marrow, hepatic Kupffer cells and red blood cells degraded by the spleen. Iron release is regulated by ferroportin, a major iron excretion protein located on the cell surface of enterocytes, macrophages and hepatocytes, which are the major cells capable of releasing iron into plasma. Hepcidin reduces its functional activity by binding to ferroportin, translocating it from the cell surface and degrading it.

The Intrinsic Hepcidin IDx ™ ELISA Kit is an ELISA test for the quantitative measurement of hepcidin in human serum and plasma.

As used herein, (a) 96 microwell plates pre-coated with anti-hepcidin antibody; (b) hepcidin-25 reference material; (c) first hepcidin-25 control; (d) second hepcidin-25 control. (E) Biotinylated hepcidin-25 tracer; (f) Streptavidin horseradish peroxidase conjugate; (g) TMB substrate; (h) Shutdown solution; (i) Wash buffer; and (j) Sample diluent , Kits are provided.

The microwell plate may be, for example, a polypropylene or polystyrene microwell plate. In some cases, the microwell strip plate is a polystyrene microwell plate. The microwell plate may be a strip plate or an integral plate.

In some cases, the kit further includes two adhesive covers for the microwell plate strip plate.

In some cases, the antibody of (a) is a monoclonal antibody having a variable heavy chain described in SEQ ID NO: 5 and a variable light chain described in SEQ ID NO: 7.

The kit may contain up to 8 vials of hepcidin-25 standard material. In some cases, the kit contains 8 vials of hepcidin-25 standard material. For example, each vial can contain 0.5 mL of hepcidin-25 standard.

In some cases, the kit contains one vial of the first hepcidin-25 control. For example, each vial of the first hepcidin-25 control can contain 0.5 mL of reagent.

In some cases, the kit contains one vial of a second hepcidin-25 control. For example, each vial of a second hepcidin-25 control can contain 0.5 mL of reagent.

The kit may include one bottle of biotinylated hepcidin-25 tracer. For example, each vial may contain 12 mL biotinylated hepcidin-25 tracer. In some cases, biotinylated hepsidin-25 tracers are (i) oxidatively folded hepsidin peptides; (ii) two (2- (2-amino-ethoxy) ethoxy) acetic acid (AEEAc) residues. A hydrophilic spacer consisting of, where the peptide of (i) is covalently linked to the hydrophilic spacer at the amino end of the peptide; and (iii) covalently to the hydrophilic spacer of (ii). Consists of linked biotin. In some cases, the hepcidin peptide has the amino acid sequence set forth in SEQ ID NO: 1.

In some cases, the kit contains one bottle of streptavidin-HRP conjugate. For example, the bottle can contain 12 mL of streptavidin-HRP conjugate.

In some cases, the kit contains one bottle of TMB substrate. For example, a bottle can contain 12 mL of TMB substrate.

In some cases, the kit contains one bottle of stop fluid. For example, a bottle can contain 12 mL of stop solution.

In some cases, the kit contains one bottle of wash buffer. For example, the bottle can contain 25 mL wash buffer. In certain cases, the kit can include a wash buffer of 20x solution, where the wash solution is diluted prior to use in the kit.

In some cases, the kit contains one bottle of sample diluent. For example, the bottle can contain 3 mL of sample diluent.

In some aspects, the kit further includes instructions for use.

Instructions may include, for example, identification of biological samples for use in the kit.

The kit may further include one or more collection means for the collection of biological samples. For example, one or more collection means can include syringes, needles, cups, swabs, or combinations thereof. Other collection means are known in the art and are contemplated herein.

In some aspects, biological samples include blood samples, tissue samples or uric acid samples. The instructions may also include instructions for processing the biological sample before use in the kit. Blood can be processed using conventional methods. For example, heparin or EDTA can be added to the blood sample. Serum is also obtained from blood samples and can be used in the assay kits described. Blood can be centrifuged to remove cellular components.

In some aspects, the kit further includes a label.

The kit can be used to detect one or more diseases or disorders associated with elevated levels of hepcidin, decreased levels of hepcidin, increased levels of iron, decreased levels of iron, or a combination thereof. it can. Non-limiting examples of diseases or disorders associated with elevated levels of hemochromatosis, decreased levels of hemochromatosis, increased levels of iron, decreased levels of iron, or combinations thereof, but not limited to Africa. Iron overload, alpha-salasemia, Alzheimer's disease, anemia, anemia due to cancer, anemia due to chronic diseases, anemia due to inflammation, arteriosclerosis or atherosclerosis, ataxia, iron-related ataxia, atransferrinemia, Cancer, celluloplasmin deficiency, chemotherapy-induced anemia, end-stage renal disease or chronic renal disease including chronic renal / renal failure, acute renal injury (AKI), liver cirrhosis, classical hemochromatosis, collagen-induced arthritis ( CIA), congenital hemochromatosis, congestive heart failure, Crohn's disease, Celiac's disease, inflammatory bowel disease (IBD), diabetes, abnormal iron distribution, abnormal iron constancy, abnormal iron metabolism, ferropotin's disease , Ferropatin mutant hemochromatosis, folic acid deficiency, Friedrich ataxia, cord myelopathy, gracile syndrome, bacterial infection, Hallerholden-Spats' disease, hemochromatosis, hemochromatosis caused by mutation of transferrin receptor 2, Hemochromatosis, hepatitis, hepatocellular carcinoma, hereditary hemochromatosis, viral infection, Huntington's disease, hyperferritinemia, hypopigmented microcytic anemia, hypoironemia, insulin resistance, iron deficiency anemia, iron deficiency , Iron overload, iron deficiency due to excess hepsidin, juvenile hemochromatosis (HFE2), multiple sclerosis, transferrin receptor 2, HFE, hemojuvelin, ferroportin, TMPRSS6 (IRIDA) or other iron metabolism Gene mutations, neonatal hemochromatosis, iron-related neurodegenerative diseases, bone loss, osteoporosis pancreatitis, pantothenate kinase-related neurodegeneration, Parkinson's disease, peragra, phagocytosis, porphyrinosis, late-onset cutaneous porphyrinosis, Pseudoencephalitis, pulmonary hemochromatosis, erythrocyte disorder, rheumatoid arthritis, septicemia, iron blast anemia, systemic erythmatodes, salacemia, intermediate salacemia, infusion iron overload, tumor, angiitis, vitamin B6 deficiency, vitamin Includes B12 deficiency, Wilson's disease, or a combination thereof.
[Invention 1001]
(a) 96 microwell strip plates pre-coated with anti-hepcidin antibody;
(b) Hepcidin-25 standard substance;
(c) First hepcidin-25 control;
(d) Second hepcidin-25 control;
(e) Biotinylated hepcidin-25 tracer;
(f) Streptavidin horseradish peroxidase conjugate;
(g) 3,3', 5,5'-Tetramethylbenzidine (TMB) substrate;
(h) Stop solution;
(i) Wash buffer; and
(j) Sample diluent
Including, kit.
[Invention 1002]
The kit of the present invention 1001 in which the microwell strip plate is a polystyrene microwell strip plate.
[Invention 1003]
The kit of the present invention 1001 further comprising two adhesive covers for the microwell strip plate.
[Invention 1004]
The kit of 1001 of the present invention, wherein the antibody of (a) is an antibody having a variable heavy chain described in SEQ ID NO: 5 and a variable light chain described in SEQ ID NO: 7.
[Invention 1005]
Kit of the present invention 1001 containing 8 vials of hepcidin-25 standard material.
[Invention 1006]
Kit of the present invention 1005, where each vial contains 0.5 mL of hepcidin-25 standard material.
[Invention 1007]
The kit of the present invention 1001 containing 1 vial of 1st hepcidin-25 control.
[Invention 1008]
The kit of 1007 of the present invention, wherein the first hepcidin-25 control vial contains 0.5 mL of reagent.
[Invention 1009]
The kit of the present invention 1001 containing 1 vial of a second hepcidin-25 control.
[Invention 1010]
The kit of 1009 of the present invention, wherein the second hepcidin 25 control vial contains 0.5 mL of reagent.
[Invention 1011]
The kit of the present invention 1001 containing one bottle of biotinylated hepcidin-25 tracer.
[Invention 1012]
The kit of the present invention 1011, wherein the vial contains a 12 mL biotinylated hepcidin-25 tracer.
[Invention 1013]
The biotinylated hepsidin-25 tracer is (i) an oxidatively folded hepsidin peptide; (ii) a hydrophilic spacer consisting of two (2- (2-amino-ethoxy) ethoxy) acetic acid (AEEAc) residues. There is a hydrophilic spacer in which the peptide of (i) is covalently linked to the hydrophilic spacer at the amino terminus of the peptide; and (iii) is covalently linked to the hydrophilic spacer of (ii). The kit of the present invention 1001 consisting of the peptide.
[Invention 1014]
The kit of the present invention 1013, wherein the biotinylated hepcidin-25 tracer comprises a hepcidin peptide having the amino acid sequence described in SEQ ID NO: 1.
[Invention 1015]
The kit of the present invention 1001 containing one bottle of streptavidin-HRP conjugate.
[Invention 1016]
The kit of the present invention 1015 containing a 12 mL bottle of streptavidin-HRP conjugate.
[Invention 1017]
The kit of the present invention 1001 containing one bottle of streptavidin-HRP conjugate.
[Invention 1018]
The kit of the present invention 1017 containing a 12 mL bottle of streptavidin-HRP conjugate.
[Invention 1019]
The kit of the present invention 1001 containing one bottle of TMB substrate.
[Invention 1020]
The kit of the present invention 1019, wherein the bottle contains 12 mL of TMB substrate.
[Invention 1021]
The kit of the present invention 1001 containing one bottle of stop solution.
[Invention 1022]
The kit of 1021 of the present invention, wherein the bottle contains 12 mL of stop solution.
[Invention 1023]
The kit of the present invention 1001 containing 1 bottle of wash buffer.
[1024 of the present invention]
The kit of the present invention 1023, wherein the bottle contains 25 mL of wash buffer.
[Invention 1025]
The kit of the present invention 1023, which comprises a wash buffer of 20 × solution.
[Invention 1026]
The kit of the present invention 1025, in which the cleaning solution is diluted before use in the kit.
[Invention 1027]
The kit of the present invention 1001 containing one bottle of sample diluent.
[Invention 1028]
The kit of the present invention 1027, wherein the bottle contains 3 mL of sample diluent.
[Invention 1029]
The kit of any of 1001-1028 of the present invention, further including instructions for use.
[Invention 1030]
The kit of the present invention 1029, the instructions comprising identifying a biological sample for use in the kit.
[Invention 1031]
The kit of the present invention 1030, further comprising one or more collection means for the collection of biological samples.
[Invention 1032]
The kit of 1031 of the present invention, wherein one or more collection means comprises a syringe, a needle, a cup, a swab, or a combination thereof.
[Invention 1033]
The kit of the present invention 1030, wherein the biological sample comprises a blood sample, a tissue sample or a urine sample.
[Invention 1034]
The kit of any of 1001 to 1033 of the present invention, further comprising a label.
[Invention 1035]
The present invention 1034 for use in the detection of one or more diseases or disorders associated with elevated levels of hepcidin, decreased levels of hepcidin, increased levels of iron, decreased levels of iron, or a combination thereof. Kit.
[Invention 1036]
One or more diseases or disorders associated with elevated levels of hemochromatosis, decreased levels of hemochromatosis, increased levels of iron, decreased levels of iron, or a combination thereof, have been associated with African iron overload, alpha salasemia, Alzheimer's disease, anemia, anemia due to cancer, anemia due to chronic diseases, anemia due to inflammation, arteriosclerosis or atherosclerosis, ataxia, iron-related ataxia, atransferrinemia, cancer, celluloplasmin deficiency, chemotherapy Induced anemia, chronic renal disease including end-stage renal disease or chronic renal / renal failure, acute renal injury (AKI), liver cirrhosis, classical hemochromatosis, collagen-induced arthritis (CIA), congenital erythropoiesis Sexual anemia, congestive heart failure, Crohn's disease, Celiac's disease, inflammatory bowel disease (IBD), diabetes, abnormal iron distribution, abnormal iron constancy, abnormal iron metabolism, ferropotin's disease, ferropotin mutant hemochromatosis, folic acid Deficiency, Friedrich ataxia, cord myelopathy, gracile syndrome, bacterial infection, Hallerholden-Spats' disease, hemochromatosis, hemochromatosis caused by mutations in transferrin receptor 2, hemochromatosis, hepatitis, hepatocytes Due to cancer, hereditary hemochromatosis, viral infection, Huntington's disease, hyperferritinemia, hypopigmental microcytic anemia, hypoironemia, insulin resistance, iron deficiency anemia, iron deficiency, iron overload, hepsidin excess Iron deficiency, juvenile hemochromatosis (HFE2), multiple sclerosis, transferrin receptor 2, HFE, hemojuvelin, ferroportin, TMPRSS6 (IRIDA), or mutations in other genes of iron metabolism, neonatal hemochromatosis Hemochromatosis, iron-related neurodegenerative diseases, bone loss, osteoporosis pancreatitis, pantothenate kinase-related neurodegeneration, Parkinson's disease, peragra, heterophagia, porphyrinosis, late-onset cutaneous porphyrinosis, pseudoencephalitis, lung Hemochromatosis, erythrocytosis, rheumatoid arthritis, sepsis, iron blast anemia, systemic erythmatodes, salacemia, intermediate salacemia, infusion iron overload, tumors, vasculitis, vitamin B6 deficiency, vitamin B12 deficiency, Wilson's disease, or The kit of the present invention 1035 which is a combination thereof.

Reference Incorporation All publications, patents, and patent applications referred to herein are specifically and individually indicated that each individual publication, patent, or patent application is incorporated by reference. To the same extent, it is incorporated herein by reference.

The novel features of the present invention are shown in detail in the appended claims. A better understanding of the features and benefits of the present disclosure is obtained by the following detailed description showing exemplary embodiments in which the principles of disclosure are used, and by reference to the accompanying drawings:
Human blood samples collected and prepared as lithium heparin (Li-heparin) and measured using the Intrinsic Life Sciences (ILS) hepcidin IDx ™ ELISA kit and the DRG hepcidin-25 Bioactive ELISA kit (n = 8). The association between hepcidin concentrations of is illustrated. The association between the hepcidin concentration of the DRG® hepcidin-25 Bioactive ELISA kit standard and the hepcidin concentration of the standard in the ILS hepcidin IDx ™ ELISA kit is illustrated. By the ILS Hepcidin IDx® ELISA kit, the DRG® hepcidin standard contained in the DRG® hepcidin-25 Bioactive ELISA kit is described in the DRG® manufacturer's instructions. It was determined to contain approximately 10 times less hepcidin than the one it had. The association between the hepcidin concentration of the ILS hepcidin IDx ™ ELISA kit standard and the hepcidin concentration of the standard substance contained in the DRG hepcidin-25 Bioactive ELISA kit is illustrated. The DRG® Hepcidin-25 Bioactive ELISA Kit measures approximately 10 times more hepcidin in the ILS Hepcidin IDx® ELISA Kit Standards than the DRG® Hepcidin-25 Bioactive ELISA Kit Standards. The result of is confirmed the data of Fig. 2. FIG. 4A shows hepsidine in a human blood sample (n = 8) collected and prepared as EDTA plasma (broken line) or lithium heparin (Li-heparin, solid line) or serum and measured by the ILS hepsidine IDx ™ ELISA kit. The relationship between concentrations is illustrated. Figure 4B shows a human blood sample (n = 8) collected and prepared as EDTA plasma (broken line) or lithium heparin (Li-heparin, solid line) or serum and measured by the DRG® hepsidin-25 Bioactive ELISA kit. The association between heparin concentrations in is illustrated.

Detailed Description of the Invention Accuracy and accuracy fall among the most important functional features of any assay. Accuracy is difficult to assess if the assay is poorly reproducible and / or if the reference material is not at the concentration disclosed in the kit. The ability to reproduce per-well and per-assay sample measurements in a single assay is the first step in understanding the feasibility of the assay. Commercially available immunoassays generally have an intraassay and interassay coefficient of variation (CV) of less than 20%. The assays and kits described in this application are significantly improved compared to this percentage.

Hepcidin is a 25-amino acid hormone and a major regulator of iron homeostasis (metabolism) in humans. Hepcidin regulates the absorption of dietary iron from the duodenum, regulates the reuse of aging red blood cell iron by macrophages, and controls the transport of iron from hepatocytes into plasma for blood production. Hepcidin is positively regulated by plasma iron and IL-6 (inflammation, infection) and suppressed by erythropoiesis via erythroferon. Abnormally low serum hepcidin is associated with iron deficiency anemia (IDA) and hereditary hemochromatosis, and high serum hepcidin is associated with chronic kidney disease (CKD), rheumatoid arthritis (RA), cancer, and iron refractory iron deficiency. It can lead to iron sequestration and inflammatory anemia (anemia due to chronic disease) observed in sexual anemia (IRIDA). Recently, hepcidin has been shown to be useful in predicting response to oral iron therapy based on hepcidin levels, which is superior to both ferritin and percent (%) transferrin saturation (% Tsat) for this application. ..

Hepcidin is involved in the regulation of iron homeostasis. Hepcidin reduces its functional activity by binding to ferroportin, translocating it from the cell surface and degrading it.

High levels of human hepcidin can result in reduced iron levels and vice versa. Mutations in the hepcidin gene that result in a lack of hepcidin activity are associated with juvenile hemochromatosis, severe iron overload. Studies in mice have revealed the role of hepcidin in the regulation of normal iron homeostasis.

Hepcidin may also be involved in iron zequestration during inflammation. It has been observed that hepcidin gene expression is strongly upregulated after inflammatory stimuli such as infection and induces an acute phase response of the vertebrate innate immune system. Hepsidin gene expression may be upregulated by lipopolysaccharide (LPS), terpentine, Freund's complete adjuvant, Freund's incomplete adjuvant, adenovirus infection and the inflammatory cytokine interleukin-6 (IL-6). A strong correlation between hepcidin expression and inflammatory anemia was also found in patients with chronic inflammatory diseases, including bacterial, fungal and viral infections.

Human hepcidin is a 25 amino acid peptide with antibacterial and iron regulatory activity. It is also called LEAP-1 (liver-expressed antimicrobial peptide). Subsequently, hepcidin cDNA encoding an 83 amino acid prepropeptide in mice and an 84 amino acid prepropeptide in rats and humans was identified in the search for iron-regulated liver-specific genes. The 24-residue N-terminal signal peptide is first cleaved to produce prohepcidin, which is then further processed to produce mature hepcidin found in both blood and urine. In human urine, the predominant form contains 25 amino acids, but in certain diseases shorter 22 and 20 amino acid peptides are also present in undetectable or very low concentrations.

The kits described herein can be used to detect the levels of mature human hepcidin in biological samples.

As used herein, "about" is generally ± 0.5%, ± 1%, ± 1.5%, ± 2%, ± 2.5%, ± 3%, ± 3.5%, ± 4%, ± 5.5% of the displayed value. , ± 6%, ± 6.5%, ± 7%, ± 7.5%, ± 8%, ± 8.5%, ± 9%, ± 9.5%, ± 10%, ± 11%, ± 12%, ± 13%, ± Means a range over the displayed values up to 14%, ± 15%, ± 16%, ± 17%, ± 18%, ± 19%, or ± 20%, or any value between them.

antibody
In one aspect, there is provided herein an antibody that specifically binds to a hepcidin or a mature human hepcidin peptide, comprising a heavy chain variable region and a light chain variable region. In some embodiments, the antibody binds to the N-terminus of a mature human hepcidin 25 amino acid peptide.

In one example, the antibodies for use in the kits provided herein are a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 5, and a light chain variable containing the amino acid sequence of SEQ ID NO: 7. Includes area.

In one aspect, the antibodies provided herein include IgG1 or IgG4 variable heavy chains and variable light chains.

The antibodies described herein are about 1 to about 500 pM, about 1 to about 10 pM, about 10 to about 20 pM, about 1 to about 29 pM, about 30 to about 40 pM, about 10 to about 100. It can have _{a dissociation constant (K d} ) of pM, or about 20 to about 500 pM.

The antibodies described herein are less than about 500 pM, less than about 450 pM, less than about 400 pM, less than about 350 pM, less than about 300 pM, less than about 250 pM, less than about 200 pM, less than about 150 pM, Less than about 100 pM, less than about 75 pM, less than about 50 pM, less than about 30 pM, less than about 25 pM, less than about 20 pM, less than about 18 pM, less than about 15 pM, less than about 10 pM, less than about 7.5 pM, It can have _{a dissociation constant (K d} ) of less than about 5 pM, less than about 2.5 pM, or less than about 1 pM.

The antibodies described herein are about 10 ^-9 to about 10 ^-14 , about 10 ^-10 to about 10 ^-14 , about 10 ^-11 to about 10 ^-14 , about 10 ^-12 to about 10 ^-14 , About 10 ^-13 to about 10 ^-14 , about 10 ^-10 to about 10 ^-11 , about 10 ^-11 to about 10 ^-12 , about 10 ^-12 to about 10 ^-13 , or 10 ^-13 to about 10 ^-14 It may have an affinity for hepcidin peptides.

The Intrinsic Hepcidin IDx ™ ELISA kit binds to the N-terminus of hepcidin-25 with high affinity (approximately 5.16 × 10 ^-11 ), a monoclonal antibody (mAb) required for bioactivity and binding to ferroportin. Competitive binding assay based on. This antibody also binds to the N-terminal isomer of low content hepcidin-25 with low affinity. Intrinsic LifeSciences has developed a described ELISA trial for hepcidin that has the ability to measure clinical levels of hepcidin in critical clinical iron disorders.

Provided herein is a composition comprising the antibodies described herein and an acceptable buffer.

As used herein, the terms "hypervariable region" and "CDR" refer to the amino acid residues of an antibody responsible for antigen binding. The CDRs contain amino acid residues from three sequence regions that complementarily bind to the antigen and are known as CDR1, CDR2, and CDR3 of the _{V H} and V _{L chains, respectively.} In the light chain variable domain, CDRs are typically approximately residue 24, according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Corresponding to ~ 34 (CDRL1), 50 ~ 56 (CDRL2) and 89 ~ 97 (CDRL3), in heavy chain variable domains, CDRs are typically approximately residues 31 ~ 35 (CDRH1), 50 ~ 65 ( Corresponds to CDRH2) and 95-102 (CDRH3). It is understood that the CDRs of different antibodies may involve insertions, and thus the amino acid numbering may differ. The Kabat numbering system utilizes the letters attached to specific residues (eg, 27A, 27B, 27C, 27D, 27E, and 27F of light chain CDRL1) to reflect arbitrary insertions in numbering between different antibodies. Numbering schemes describe such insertions. Alternatively, in the light chain variable domain, CDRs are typically according to Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987), approximately residues 26-32 (CDRL1), 50-52 ( Corresponding to CDRL2) and 91-96 (CDRL3), and in the heavy chain variable domain, the CDRs are typically approximately residues 26-32 (CDRH1), 53-55 (CDRH2) and 96-101 (CDRH3). Corresponds to.

As used herein, "framework region" or "FR" refers to a framework amino acid residue that forms part of an antigen-binding pocket or antigen-binding groove. In some embodiments, the framework residues form a loop that is part of the antigen binding pocket or antigen binding groove, and the amino acid residues in the loop may or may not contact the antigen. Framework areas generally include areas between CDRs. In the light chain variable domain, FR is typically approximately residue 0, according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Corresponding to ~ 23 (FRL1), 35 ~ 49 (FRL2), 57 ~ 88 (FRL3), and 98 ~ 109, in the heavy chain variable domain, FR is typically approximately residue 0-30 (FRH1). , 36-49 (FRH2), 66-94 (FRH3), and 103-133. As mentioned above by Kabat numbering of the light chain, insertion of the heavy chain is described in a similar manner (eg, 35A, 35B of CDRH1 of the heavy chain). Alternatively, in the light chain variable domain, FR is typically approximately residues 0-25 (FRL1), 33-49 (according to Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987)). Corresponding to FRL2), 53-90 (FRL3), and 97-109 (FRL4), in the heavy chain variable domain, FR is typically approximately residues 0-25 (FRH1), 33-52 (FRH2). , 56-95 (FRH3), and 102-113 (FRH4).

The loop amino acids of FR can be evaluated and determined by examining the three-dimensional structure of the antibody heavy chain and / or the antibody light chain. Tertiary structure can be analyzed for solvent-contactable amino acid positions, as solvent-contactable amino acid positions can form loops and / or result in antigen contact in the antibody variable domain. Some of the solvent-contactable positions can tolerate amino acid sequence diversity, while others (eg, structural positions) are generally less diverse. The three-dimensional structure of the antibody variable domain can be derived from crystal structure or protein modeling.

The constant domain (Fc) of an antibody is not directly involved in binding the antibody to the antigen, but rather varies, for example, the involvement of the antibody in antibody-dependent cellular cytotoxicity through interaction with the Fc receptor (FcR), etc. It exhibits an effective effector function. The Fc domain can also increase the bioavailability of the antibody in the circulation after administration to the subject.

The term "specific" refers to a situation in which an antibody does not exhibit any significant binding to molecules other than the antigen containing the epitope recognized by the antibody. The term is also applicable, for example, when the antigen-binding domain is specific for a particular epitope carried by several antigens, in which case an antibody or antigen-binding fragment thereof carrying the antigen-binding domain. Has the ability to bind to various antigens carrying the epitope. The term "preferentially binds" or "specifically binds" means that an antibody or fragment thereof binds to an epitope with a higher affinity than it binds to an irrelevant amino acid sequence, and other polys containing the epitope. Having cross-reactivity to peptides means that they are not toxic at the level at which they are formulated for administration to human applications. In one aspect, such affinity is at least 1-fold higher, at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher than the affinity of the antibody or fragment thereof for an irrelevant amino acid sequence. At least 6 times higher, at least 7 times higher, at least 8 times higher, at least 9 times higher, 10 times higher, at least 20 times higher, at least 30 times higher, at least 40 times higher, at least 50 times higher, at least 60 times higher, at least 70 times higher, at least 80 times higher, at least 90 times higher, at least 100 times higher, or at least 1000 times higher. The terms "immune-reactive," "binding," "preferentially binding," and "specifically binding" are used interchangeably herein. The term "bond" refers to a direct association between two molecules, for example, by covalent interactions, electrostatic interactions, hydrophobic interactions, and ionic and / or hydrogen bond interactions under physiological conditions. Refers to interactions such as salt bridges and water bridges, as well as conventional means of any other bond.

When applied to an antibody, "isolated" (used interchangeably with "substantially pure" or "purified") is either chemically synthesized or expressed in host cells. , Means an antibody purified to remove associated and contaminating proteins. The term generally means a polypeptide that has been separated from other proteins and nucleic acids that naturally occur with it, and / or the substance used to purify it. In some cases, the antibodies provided in the kits described herein are about 95%, about 96%, about 97%, about 98%, about 99% or more pure.

のアミノ酸配列を有する。酸化的にフォールディングされるヘプシジンペプチドはヒトヘプシジン25のアミノ酸配列を有しうる。 Tracer As used herein, (i) an oxidatively folded hepcidin peptide; (ii) a hydrophilic spacer consisting of two (2- (2-amino-ethoxy) ethoxy) acetic acid (AEEAc) residues, wherein , (I) peptide is covalently linked to the hydrophilic spacer at the amino-terminus (N-terminus) of the peptide; and (iii) biotin covalently linked to the hydrophilic spacer of (ii). An immunoassay tracer reagent comprising. In some cases, the hepcidin peptide is oxidatively folded before being covalently linked to the hydrophilic spacer of (ii). In some cases, hepcidin peptides that are oxidatively folded

Has an amino acid sequence of. An oxidatively folded hepcidin peptide can have the amino acid sequence of human hepcidin 25.

The hepsidin peptide is a step of (a) solubilizing hepsidin with an acetic acid solution to produce a first solution; (b) diluting the first solution with an aqueous buffer solution containing a chaotropic reagent, an organic alcohol, and an oxidation reagent. , The step of producing a second solution; and (c) the step of adjusting the pH of the second solution to a level between about 5 and 7 can be oxidatively folded. The organic alcohol may be approximately 10% isopropyl alcohol; (ii) the pH may be adjusted by the addition of ammonium hydroxide; or (iii) oxidation may take place at room temperature.

Kits Provided herein are kits that include antibodies, acceptable carriers and excipients, the compositions described herein, and labels that describe the steps for using the kits. In some cases, the kit may also include instructions for obtaining and processing the sample prior to use in the kit. In one aspect, herein, a 96-well microwell plate coated with an antibody and blocked with a blocking agent as described herein, a biotinylated tracer molecule, streptavidin horseradish peroxidase, a substrate solution, Kits are provided for diagnosing disorders associated with elevated hepsidin levels or abnormal iron homeostasis, including stop solutions, wash buffers, sample diluents, and reference materials (ie, positive controls). Diluted solutions containing no reference material can be used as a negative control.

Provided herein are container means comprising the compositions described herein. Container means can be any suitable container capable of containing liquid or lyophilized compositions, including but not limited to vials, syringes, bottles, intravenous (IV) bags or ampoules. The container means can be made of any material suitable for storing the contents of the kit. Vessel means are not limited to these, but are about 0.1 ml to about 50 ml or more; for example, about 0.5 ml, about 1 ml, about 2 ml, about 5 ml, about 10 ml, about 15 ml, about 20. Suitable for use in the methods described herein, including ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml or more, or any integer between them. It may have the ability to hold any volume of liquid. In some cases, the container means holds the solid or liquid composition. If the container means retains the solid composition, the container means is limited to, but not limited to, about 0.01 mg to about 50 g or more; for example, about 0.5 mg, about 1 mg, about 2 mg, about 5 mg. , About 10 mg, about 15 mg or more, or any integer in between, and may have the ability to hold any amount of composition for use in the methods described herein. ..

The kit may also include means for containing reagents under hermetic control for commercial sale. Such a container may include a blow molded plastic container in which the desired vial is held for injection and / or in it. The kit can also include printed matter for the use of the substances in the kit.

The formulation / preparation contained in the package or kit may additionally contain a buffer, a preservative, a stabilizer, or a combination thereof. Each component of the kit can be enclosed in a separate container and all of the various containers can be in one package. The kit of the present invention can be designed for refrigerated storage or room temperature storage.

In addition, the reagent formulation / preparation may contain a stabilizer to increase the shelf life of the kit and may include, for example, bovine serum albumin (BSA). If the composition is lyophilized, the kit may further contain a preparation of the solution for reconstitution of the lyophilized preparation. Acceptable reconstruction solutions are well known in the art and include, for example, pharmaceutically acceptable phosphate buffered saline (PBS).

Packages and kits can further include one or more components for assays, such as ELISA assays. For example, the kit may include a microwell plate such as a 96-well Miklewell plate. In some cases, the microwell plates of the kits provided herein are coated with an anti-hepcidin antibody and blocked with a blocking agent prior to packaging. Such plates can be sealed in sterilized plastic packaging and vacuum sealed. Plates are prepared under sterile conditions to avoid contamination.

Packages and kits can further include one or more components for sample collection (eg, syringes, needles, cups, vials, bottles, swabs, etc.).

Packages and kits may further include, for example, a label stating the product description.

The term "packaging material" refers to the physical structure that houses the components of the kit. The packaging material can maintain the components sterilized and can be made of materials commonly used for such purposes (eg, paper, corrugated board, glass, plastic, foil, ampoules, etc.). Labels or packaging inclusions may include appropriate written instructions. As such, the kit may additionally include labels or instructions for using the kit components in any of the methods of the invention. The kit can include the compound in a pack or dispenser along with instructions for administering the compound in the methods described herein.

The instructions may include instructions for performing any of the methods described herein, including diagnostic methods.

Instructions are attached on "printed matter", eg, on paper or board paper in or attached to the kit, or on a vial or tube that is attached to or contains components of the kit or packaging material. It can be on the label. Instructions include computer-readable media such as flash / cloud drives, disks (floppy disks or hard disks), optical CDs such as CD-ROM / RAM or DVD-ROM / RAM, magnetic tape, RAM and ROM. Additional inclusions may be included in these hybrids such as electrical storage media, IC chips, and magnetic / optical storage media.

Diagnostic method
In one embodiment, the antibody or antigen binding fragment further comprises a detectable component. Detection can be done in vitro. In vitro assays for the detection and / or determination (quantitative, qualitative, etc.) of hepcidin by the antibodies described herein include, for example, competitive enzyme-linked immunosorbent assay (ELISA). In vitro detection, diagnosis or monitoring of hepsidin can be performed by obtaining a sample from a subject (eg, a blood sample, tissue sample or urine sample) and, for example, in a competitive ELISA assay as described in the examples below. This can be done by testing the sample.

As used herein, (a) biological samples from subjects suspected of having hepcidin-related disorders under conditions that allow binding of the antibody or antigen-binding fragment thereof to hepcidin, the antibodies described herein or The step of contacting with the antigen-binding fragment; and (b) the step of detecting and / or quantifying the antibody or hepcidin bound to the antigen-binding fragment in the sample quantified above the threshold level (b). A method of diagnosing hepcidin-related disorders, comprising steps, wherein the amount of hepcidin in hepcidin indicates the presence of hepcidin-related disorder and the amount below the threshold level indicates the absence of hepcidin-related disorder is provided. In some cases, biological samples are processed / processed prior to use in the described kit assays. For example, EDTA or heparin can be added to the sample. Blood can also be centrifuged to obtain serum for testing.

(a) Biological samples from humans suspected of having an inflammatory disease under conditions that allow the antibody or its antigen-binding fragment to bind to hepcidin with the antibody or antigen-binding fragment thereof described herein. Contacting; and (b) the step of detecting and / or quantifying hepcidin bound to an antibody or antigen-binding fragment thereof, where the amount of hepcidin quantified above the threshold level (b) is an inflammatory disease. A method of distinguishing an inflammatory disease from a non-inflammatory disease, including steps, indicating the presence of, and an amount below a threshold level indicating the absence of the inflammatory disease. In some cases, biological samples are processed / processed prior to use in the described kit assays. For example, EDTA or heparin can be added to the sample. Blood can also be centrifuged to obtain serum for testing.

A "subject" according to one aspect of the application (eg, a human or non-human animal, eg, a mammal such as a primate, rodent, cow, horse, pig, sheep) is a disease described herein. Or a mammal that presents with one or more clinical symptoms and / or symptoms of the disorder.

Also provided herein are diagnostic methods for detecting hepcidin-related diseases or disorders or iron-related diseases or disorders using the antibodies described herein. In certain examples, the method detects diseases or disorders associated with elevated levels of hepcidin, decreased levels of hepcidin, increased levels of iron, decreased levels of iron, or a combination thereof. Hemochromatosis-related disorders, inflammatory disorders, and iron constitutive disorders or disorders to which the method can be applied are, but are not limited to, African iron overload, alpha salacemia, Alzheimer's disease, anemia, cancer-induced anemia, chronic. Disease-induced anemia, inflammation-induced anemia, arteriosclerosis or atherosclerosis (including coronary artery disease, cerebrovascular disease or peripheral obstructive arterial disease), ataxia, iron-related ataxia, atransferase, cancer, Celluloplasmin deficiency, chemotherapy-induced anemia, end-stage renal disease or chronic renal / renal disease including chronic renal / renal failure (stage I, II, III, IV or V), acute renal injury (AKI), liver cirrhosis, classical Hemochromatosis, collagen-induced arthritis (CIA), conditions with hepsidin overload (elevated hepsidin), congenital erythropoiesis anemia, congestive heart failure, Crohn's disease, celiac disease, inflammatory bowel disease (IBD), diabetes, iron Abnormal distribution in the body, abnormal iron constancy, abnormal iron metabolism, ferropotin disease, ferropotin mutant hemochromatosis, folic acid deficiency, Friedrich ataxia, cord myelopathy, Gracil syndrome, H. pylori infection or Other bacterial infections, Hallerholden-Spats disease, hemochromatosis, hemochromatosis caused by mutations in transferrin receptor 2, hemoglobin dysfunction, hepatitis, hepatitis (Brock), hepatitis C, hepatocellular carcinoma, hepsidin deficiency, hereditary Hemochromatosis, HIV or other viral diseases, Huntington's disease, hyperferritinemia, hypopigmental microcytic anemia, hypoironemia, insulin resistance, iron deficiency anemia, iron deficiency, iron overload, hepsidin excess Iron deficiency due to iron deficiency, juvenile hemochromatosis (HFE2), multiple sclerosis, transferrin receptor 2, HFE, hemochromatosis, ferroportin, TMPRSS6 (IRIDA) or other gene mutations in iron metabolism, neonatal hemochromatosis, iron Related to neurodegenerative diseases, bone loss, osteoporosis pancreatitis, pantothenate kinase related neurodegeneration, Parkinson's disease, peragra, phagocytosis, porphyrinosis, late skin porphyrinosis, pseudoencephalitis, pulmonary hemochromatosis, erythrocyte disorder Includes, rheumatoid arthritis, sepsis, iron blast anemia, systemic erythroidosis, salacemia, intermediate salacemia, infusion iron overload, tumors, vasculitis, vitamin B6 deficiency, vitamin B12 deficiency, and / or Wilson's disease To.

ヒトヘプシジン-20ペプチド(ヘプシジン-20、hep-20、Hep-25、hヘプシジン-20)：(20aa)

ヒトヘプシジン22ペプチド(ヘプシジン-22、hep-22、Hep-22、hヘプシジン-22):(22aa)

Sequence Human hepcidin peptide (hepcidin-25, hep-25, Hep-25, h hepcidin-25): (25aa)

Human hepcidin-20 peptide (hepcidin-20, hep-20, Hep-25, h hepcidin-20): (20aa)

Human hepcidin 22 peptide (hepcidin-22, hep-22, Hep-22, h hepcidin-22): (22aa)

583 VHアミノ酸配列：

583 VL核酸配列：

583 VLアミノ酸配列：

Nucleic acid sequence and amino acid sequence of variable heavy chain and variable light chain of hepcidin MAb 583.
583VH Nucleic Acid Sequence:

583 VH amino acid sequence:

583 VL Nucleic Acid Sequence:

583 VL amino acid sequence:

可変軽鎖CDR

CDR-1, CDR-2, and CDR-3 polynucleotide sequences of the variable heavy and variable light chains of hepcidin MAb 583.
Variable heavy chain CDR

Variable light chain CDR

The application can be better understood by reference to the following non-limiting examples provided as exemplary embodiments of the application. The following examples are presented to better illustrate the embodiments, but should not be construed at all as limiting the broad scope of the application.

Example 1: Intrinsic hepcidin IDx ™ ELISA
Intrinsic Life Sciences (ILS) hepcidin IDx ™ ELISA kit provides bioactive bioactivation of hepcidin-25 in test specimens to a certain number of high-affinity anti-hepcidin-25 N-terminal specific mAb binding sites. Competitive binding assay with human hepcidin-25 tracer. First, purified hepcidin-25 standard, hepcidin-25 controls (control 1 and control 2), and patient samples (all repeated twice) are added to a mAb-coated microwell strip plate and biotinylated hepcidin-25. Incubated with tracer for 60 minutes. The biotinylated hepcidin-25 tracer competes with native hepcidin or reference hepcidin for a fixed number of N-terminal specific antibody binding sites. Thus, the amount of bound biotinylated hepcidin-25 tracer gradually decreases as the concentration of bound native serum hepcidin from the patient sample increases. The unbound biotinylated hepcidin-25 tracer is washed away and a streptavidin horseradish peroxidase (HRP) conjugate is added to the wells. Wells are incubated for 30 minutes and washed to remove unbound streptavidin-HRP. Add the TMB substrate for 15 minutes and stop the reaction by adding a stop solution. Absorbance at 450 nm was measured using a microwell plate reader and the data were recorded.

Materials not provided Distilled or deionized water

Precision pipettes (single channel and multichannel), disposable pipette tips (100 μl and 1000 μl)

Microwell plate reader, desktop centrifuge capable of reading absorbance at 450 nm.

Flat head vortex mixer, microplate shaker, absorbent paper.

Curve fitting software, graph paper.

Collection and Handling of Specimens Blood samples can be collected in serum, serum separators, lithium or sodium heparin plasma, or EDTA plasma tubes. Serum and plasma samples can be refrigerated at 2-8 ° C for up to 24 hours from the time of blood collection.

If storage for more than 24 hours is required, plasma or serum can be cryopreserved at -20 ° C to -80 ° C for up to 1 year. Avoid multiple freeze-thaw cycles.

Prior to the assay, frozen serum or plasma should reach room temperature. If visible precipitates are present, the samples are gently mixed and centrifuged prior to use. Do not use hemolyzed, contaminated or seborrheic samples.

Reagent Preparation All reagents and specimens must be allowed to reach room temperature before use. All reagents should be mixed gently without foaming. Once the procedure is initiated, all steps should be completed without interruption. Prepare 1 x wash buffer by adding 475 mL of distilled or deionized water to the contents of the bottle (25 mL, 20 x). Store wash buffer at room temperature (18-24 ° C).

Standard Assay Techniques For best results, premix samples, reference materials, controls and tracers for ELISA in 96-well polypropylene (PP) plates provided in the kit.

Using a multichannel pipette, transfer 110 μl of biotinylated hepcidin-25 tracer to the required wells of the PP plate.

Transfer each of the 22 μl hepcidin reference material, sample and each hepcidin control to a PP plate. If the patient sample requires further dilution, see the instructions for diluting the sample below.

Carefully mix the solution using a multi-channel pipette. Transfer 120 μl of this solution from the PP plate to the corresponding well in a 96 microwell strip plate.

Incubate the microwell strip plate at room temperature for 60 minutes with stirring (350 rpm). Quickly shake the contents of the well into the waste reservoir.

Dispense 300 μl of 1 × wash buffer into each well, then quickly shake the wash buffer into the waste reservoir. Sharply hit the wells on the absorbent paper to remove any residual droplets. Repeat washing twice, a total of three times.

Dispense 100 μl of streptavidin-HRP conjugate into each well.

Incubate for 30 minutes at room temperature with stirring (350 rpm). Quickly shake the contents of the well into the waste reservoir.

Dispense 300 μl of 1 × wash buffer into each well, then quickly shake the wash buffer into the waste reservoir. Sharply hit the wells on the absorbent paper to remove any residual droplets. Repeat washing twice, a total of three times.

Dispense 100 μl of TMB substrate into each well using a multi-channel pipette. Incubate for 15 minutes at room temperature, preferably in the dark.

Dispense 50 μl of stop solution into each well to stop the enzymatic reaction. Carefully mix the plate contents for 20-30 seconds.

Read the absorbance at 450 nm within 15 minutes of adding the stop solution.

Instructions for Diluting the Sample If the hepcidin concentration in the sample approaches or exceeds the upper limit of quantification (approximately 250 ng / ml), dilute the sample and rerun ELISA (see warnings and cautions). .. Dilute with sample diluent (supplied) in a low protein binding 96 microwell plate (not included) or a separate low protein binding microcentrifuge tube (not included). See the sample dilution examples shown in the table below. Transfer 22 μl of diluted sample to a 96-well PP plate as described in the standard assay procedure (above).

When performing data calculations, always multiply the sample result by the sample dilution factor.

Any Assay Technique Bring all reagents to room temperature before performing the assay. Gently mix all reagents before use.

Dispense 20 μl of hepcidin standard, control and sample into each well.

Dispense 100 μl of biotinylated hepcidin-25 tracer into each well.

Incubation No. 1-Incubate the microwell strip plate at room temperature for 60 minutes with stirring (350 rpm). Quickly shake the contents of the well into the waste reservoir.

Dispense 1 x wash buffer of wash number 1-300 μl into each well, then quickly shake the wash buffer into the waste reservoir. Sharply hit the wells on the absorbent paper to remove any residual droplets. Repeat washing twice, a total of three times.

Dispense 100 μl of streptavidin-horseradish peroxidase (HRP) conjugate into each well.

Incubation No. 2-Incubate for 30 minutes at room temperature with stirring (350 rpm). Quickly shake the contents of the well into the waste reservoir.

Dispense 1 x wash buffer of wash number 2-300 μl into each well, then quickly shake the wash buffer into the waste reservoir. Sharply hit the wells on the absorbent paper to remove any residual droplets. Repeat washing twice, a total of three times.

Using a multi-channel pipette, dispense 100 μl of TMB (3,3', 5,5'-tetramethylbenzidine) substrate into each well.

Incubation No. 3-Incubate for 15 minutes at room temperature, preferably in the dark.

Stop-Dispens 50 μl of stop solution into each well to stop the enzymatic reaction. Carefully mix the plate contents for 20-30 seconds.

Read the absorbance at 450 nm within 15 minutes of adding the stop solution.

Result calculation Software calculation:
Create a standard curve and use either the Four Parameter logistic (4PL) or Point-to-Point curve fitting algorithm by GRAPHPAD PRISM® (Version 6.01) software (worldwide website: graphad.com). , The sample hepcidin concentration was determined. This test was developed and validated using 4PL curve fitting. Both methods produce very similar results. The inventors recommend that one method be used consistently after selection.

Manual calculation:
To build a standard curve, plot the absorbance of the hepcidin-25 standard (OD 450 nm) (vertical axis) with respect to the concentration of the hepcidin-25 standard (horizontal axis) on linear graph paper. Draw the best curve through the points. Read and record the absorbance of the control and each unknown sample from the curve.

The calculations can also be constructed using a suitable computer program.

Example of hepcidin standard material curve data

Correlation between serum heparin and plasma heparin The graph in Figure 1 shows eight human blood samples (low, medium, high heparin) obtained and prepared as serum, lithium heparin plasma, and EDTA plasma using standard techniques. Represents a regression analysis of serum heparin to plasma heparin from -25). Hepcidin-25 concentrations were measured using an Intrinsic hepcidin IDx ™ ELISA kit. Strong correlation between serum and EDTA plasma (●, y = 1.096x-1.25, R ² = 0.994) and between serum and lithium heparin plasma (■, y = 0.946x + 0.19, R ^{2 = 0.994)} Sex was observed. See Figure 1.

Storage and stability Store the kit at 2-8 ° C.

All microwell strip plates remain sealed in dry bags with desiccant.

The reagents are stable until the expiration date of the kit.

Do not expose the test reagents to heat, sun or strong light.

Warnings and Cautions Substances that may be biologically harmful: Standards and controls are human sources that have been tested with FDA-approved reagents for hepatitis B surface antigens and HIV antibodies and found to be non-responsive. Contains ingredients. However, neither test method provides complete assurance that HIV, hepatitis B virus or other infectious agents are absent. These reagents should be treated as biosafety level 2 substances.

Do not pipette by mouth. Do not smoke or drink alcohol in areas where specimens or kit reagents are handled.

The components in this kit are integrated units. Reagents from different lots should not be mixed. References, controls and patient samples are recommended to be run twice. Optimal results are obtained by adhering to this protocol.

Accurate and accurate pipette operation, careful cleaning and detoxification, and accurate timing at specified temperatures are essential. Deviations from the recommended values can result in invalid data.

Check the hepcidin-25 standard substance concentration value for each vial. This value can vary from lot to lot.

Absorbance of 250 ng / ml hepcidin-25 standard substance (standard substance 7) (A) Patient samples with A 450 nm below 450 nm should be diluted and re-executed (see instructions for diluting samples). I want to be).

Example 2: Assay Coefficient of Variation (CV) and Gender Differences in Hepcidin Accuracy and accuracy fall among the most important functional features of any assay. Accuracy is difficult to assess if the assay is poorly reproducible. The ability to reproduce per-well and per-assay sample measurements in a single assay is the first step in understanding the feasibility of the assay.

Commercially available immunoassays for hepcidin generally have an intraassay and interassay coefficient of variation (CV) of less than 20%. Our task has been to design a kit with a more accurate CV for the higher accuracy of the hepcidin detection assay. Higher accuracy measurements allow the diagnosis of conditions associated with increased or decreased levels of hepcidin with greater accuracy.

^＊平均n＝16；3回のアッセイに対するCV。^3アッセイを通してn＝48。 Serum samples containing low, medium, or high levels of endogenous hepcidin were selected. These samples were run in three independent assays (n = 16). CV was calculated for each sample in each assay. The intra-assay accuracy was calculated as the mean CV between assays, and the inter-assay accuracy was calculated as the CV of all measurements (n = 48). The mean values for the low, medium, and high samples were 22.2 ng / ml, 92.8 ng / ml, and 222.5 ng / ml, respectively. All intra-assay and inter-assay CVs identified by this method were below 5% and were very accurate. Serum samples containing low, medium, or high levels of endogenous hepcidin were measured in 3 assays (n = 16). The mean hepcidin value and the associated coefficient of variation are listed in the table below.

^* Mean n = 16; CV for 3 assays. ^ 3 n = 48 through the assay.

Confirmation of good accuracy then allows evaluation of accuracy. Spike recovery is the most direct way to assess accuracy due to the fact that the amount added is known and does not require a "gold-standard" comparative assay. Two matrices: assay buffer and serum were selected for addition. The assay buffer is a simple matrix in the absence of interfering substances. The serum sample selected is a clinically relevant complex matrix lacking endogenous hepcidin. The acceptance criteria for addition recovery is to determine the relative error (RE) ± 20% for each level tested. Hepcidin was added into each matrix at levels across the analytical range.

Addition Recovery: The two matrices selected for addition recovery are serum samples free of assay buffer and endogenous hepcidin. Hepcidin was added into these matrices over the analytical range. Relative error was calculated for each measurement. As shown in the table below, the mean relative error (RE) of buffer and serum was -12% and -4%, respectively.

The mean RE of buffer and serum was -12% and -4%, respectively.

Healthy initial blood donor data can be used to establish a reference range. References are calculated separately because male and female hepcidin levels are significantly different from each other. The mean hepcidin for females (n = 148) and males (n = 142) was determined to be 21.9 ng / ml and 41.5 ng / ml, respectively. 5% to 95% quantiles for women and men are 2.6 to 59.5 ng / ml and 7.3 to 104.3 ng /, respectively, as shown in the table below, which provides mean, median and selective quantile data. It reached ml.

Another important feature of the robust assay is dilution linearity. This involves the measurement of high hepcidin serum samples serially diluted with assay buffer. Acceptable linearity occurs when the diluted sample values result in a relative error of ± 20% compared to the undiluted sample measurements. Two high hepcidin serum samples were diluted 1: 2 and 1: 4 with assay buffer and measured in parallel with the undiluted sample. As shown in the table below, the mean relative errors of the 1: 2 and 1: 4 dilutions were -2% and -1%, respectively.

Dilution linearity: Two serum samples were diluted 1: 2 and 1: 4, and then hepcidin was measured using the ILS hepcidin IDx ™ ELISA kit, the current kit described in this patent. Relative error was calculated from comparison to undiluted measurements. Commercial tests produced relative errors of 3% and -3% for 1: 2 and 1: 4 dilutions, respectively.

The experiments and data presented above show that the ILS hepcidin IDx ™ ELISA kit provides clinical analysis and subsequent clinical analysis of patient samples for hepcidin-25 for the diagnosis of hereditary and acquired anemia and iron overload. Clearly show that it has excellent technical features and practicality for testing.

Example 3: Comparison of Current Kit with DRG® Commercial Kit Using the manufacturer's instructions, the current kit described in this application was converted to DRG® Hepcidin 25 bioactive ELISA (EIA-5258). , Version 4.1, revised April 28, 2014, compared with DRG International, Inc., USA. Simply put, the materials and protocols for the DRG® kit are:

DRG® Reagent Microtiter Wells, 12x8 (Division) Strips, 96 Wells Provided by Kit; Wells Coated with Anti-Hepsidin-25 Antibody (Monclonal).

Standard substance (standard substance 0-5), 6 vials (lyophilized), 0.2 mL; Concentration: 0-2-6.5-25-45-80 ng / mL; Conversion; 1 ng / mL = 0.358 nmol / L Non-mercury Contains system preservatives.

Control Low & High, 2 vials, (lyophilized), 0.2 mL, control values and range described on vial label or QC-datasheet.

Includes assay buffer, 1 vial, 14 mL, ready to use, non-mercury preservative.

Enzyme conjugate, 1 vial, 7 mL, ready to use, biotin-conjugated hepcidin-25; contains non-mercury preservatives.

Enzyme complex, 1 vial, 14 mL, ready to use, HRP-conjugated streptavidin; containing non-mercury preservatives.

Substrate solution, 1 vial, 14 mL, ready to use, tetramethylbenzidine (TMB).

Includes stop solution, 1 vial, 14 mL, ready to use, 0.5 M H2 SO4.

Cleaning solution, 1 vial, 30 mL (40 x concentrated).

Bring all reagents and the required number of strips to room temperature before use.

The lyophilized contents of the DRG® standard substance standard vial provided by the kit are reconstituted with 0.2 mL deionized water and allowed to stand for a minimum of 10 minutes. Mix the reference material multiple times before use.

DRG® Control Lyophilized contents are reconstituted with 0.2 mL deionized water and allowed to stand for a minimum of 10 minutes. Mix the control multiple times before use.

DRG® Cleaning Solution Add deionized water to the cleaning solution concentrated to 40x. Dilute 30 mL of concentrated wash solution with 1170 mL deionized water to a final volume of 1200 mL.

DRG® Specimen Collection and Preparation Serum or heparin plasma can be used in this assay. EDTA plasma and citrate plasma result in reduced values (about 20%).

Specimen collection Serum: Blood is collected by venipuncture (eg, Sarstedt Monovette for serum), coagulated, and separated by centrifugation at room temperature. Do not centrifuge before complete coagulation occurs. Samples containing anticoagulants may require increased coagulation time.
Plasma: Whole blood should be collected in a centrifuge tube containing an anticoagulant (eg, Sarstedt Monovette with appropriate plasma preparation) and centrifuged immediately after collection.

Specimen Dilution If the sample is found to contain more than the highest amount of reference material in the initial assay, the sample can be diluted with assay buffer and reassayed as described in the assay procedure. This dilution factor needs to be taken into account when calculating the concentration. Example :
(a) Dilution 1:10:10 μL serum + 90 μL assay buffer (completely mixed)
(b) Dilution 1: 100: 10 μL Dilution (a) 1: 10 + 90 μL Assay Buffer (Mix Completely)

Assay Techniques Each run must include a standard curve.
1. Secure the desired number of microtiter wells in the frame holder.
2. Dispense 100 μL of assay buffer into the appropriate well.
3. Dispense 20 μL of each of the standard, control and sample into the appropriate wells using a new disposable tip.
4. Dispense 50 μL of enzyme conjugate into each well. Mix thoroughly for 10 seconds. Thorough mixing is important in this process.
5. Incubate for 60 minutes at room temperature under stirring (300-700 rpm).
6. Quickly shake off the contents of the well. Rinse wells 3 times with 400 μL diluted wash solution per well (when using a plate washer) or 4 times with 300 μL diluted wash solution per well for manual washes. Sharpen the wells onto the absorbent paper to remove any residual droplets.
7. Dispense 100 μL of enzyme complex into the appropriate wells.
8. Incubate for 30 minutes at room temperature without stirring.
9. Quickly shake off the contents of the well. Rinse wells 3 times with 400 μL diluted wash solution per well (when using a plate washer) or 4 times with 300 μL diluted wash solution per well for manual washes. Sharpen the wells onto the absorbent paper to remove any residual droplets.
10. Add 100 μL of substrate solution to each well.
11. Incubate for 20 minutes at room temperature.
12. Stop the enzymatic reaction by adding 100 μL of stop solution to each well.
13. The absorbance (OD) of each well was determined by a microplate reader at 450 ± 10 nm. It is recommended to read the wells within 10 minutes after adding the stop solution.

Calculation of Results Calculate the average absorbance for each set of reference material, control and sample. Using a semi-log graph paper, construct a standard curve by plotting the average absorbance obtained from each standard substance for that concentration by the absorbance value on the vertical (Y) axis and the concentration on the horizontal (X) axis. ..

The average absorbance of each sample is used to determine the corresponding concentration from the standard curve.

Automation method: IFU results are automatically calculated using 4 PL (4 Parameter Logistics) curve fitting. 4 Parameter Logistics is the preferred method. Other data reduction functions can produce slightly different results. The concentration of the sample can be read directly from this standard curve. Samples with concentrations higher than the highest standard concentration need to be further diluted or reported as> 80 ng / mL.

Comparison of Results Between Current Kit and DRG hepcidin-25 bioactive Kit Figure 1 shows a linear regression of plasma hepcidin results from the ILS hepcidin IDx ™ ELISA kit and the DRG ™ comparison kit. Is shown. The slope of the regression indicates that the DRG® kit measures approximately 9-fold lower hepcidin concentration in HIPPA-compliant Li-heparin samples compared to the ILS hepcidin IDx® ELISA kit. The correlation between the two kits was good at ^{R 2 = 0.91.} Due to the inconsistency in plasma hepcidin concentration, the inventors measured the reference reference material from this kit using the DRG® comparison kit and used the ILS hepcidin IDx® ELISA to measure the DRG® kit. Reference reference material from was measured.

Figure 2 shows the measurement of DRG® comparative reference reference material with the standard material of this kit. The results of the comparison of reference materials are shown in Figure 2 as a linear regression between this kit and the DRG® comparison kit. The regression is explained by Equation y = 0.1101x−0.1176, R ² = 0.9907. While the results are highly correlated between the kit and the DRG kit, it is clear that the DRG® reference material is approximately 9 times lower than that from the present invention for standards of the same concentration. Shown.

Figure 3 compares reference reference materials from this kit as measured using the DRG® comparison kit. The result of this comparison is shown in FIG. 3 as a linear regression explained by Eq. y = 10.648x −4.3862, R ^{2 = 0.9899.} This comparison confirms that the DRG® reference reference material is 10.6 times lower than that labeled on the reference standard material described in the DRG® kit. The reference material used in this kit contains the actual concentration of hepcidin labeled and the DRG® kit needs to be tuned to produce results similar to the MALDI-TOF Mass Spec assay. Indicates that a reference reference material is used.

Figures 4A and 4B demonstrate that this kit is an improvement over existing commercial DRG® kits. These figures show the relative effects of the sample matrix on each hepcidin ELISA. FIG. 4A shows the results of this kit from the same blood sample prepared as serum against the same sample prepared as plasma using Li-heparin blood tube or K _{2-EDTA, respectively.} Samples prepared as plasma with either Li-heparin or K2-EDTA by linear regression inspection produced very similar slopes in both plasma matrices, with a serum sample slope of approximately ± 10%. There is.

FIG. 4B shows the same sample prepared as serum or Li-heparin or K _2-EDTA. Regression analysis and formulas for both Li-heparin and K _2- EDTA show a much higher effect (± 34%) of sample matrix type on serum compared to the kit (Li-heparin; y = 0.7915x). -0.1571; K _2- EDTA; y = 0.6545x-0.529). The higher agreement between plasma and serum hepcidin concentrations in this kit is a clear improvement over DRG®.

In conclusion, the kits of the invention are significantly more than the data contained in the comparative DRG® hepcidin-25 (Bioactive) ELISA kit (EIA-5258) and the new version of DRG® (EIA-5782). It is a technical improvement.

The kit utilizes a reference reference material that is formulated at the concentration indicated on the reference reference material tube in the kit and that is not artificially adjusted to reproduce the results of the MALDI-TOF MS test. As such, such artificial preparations can carry the significant pre-analysis error itself (Figures 2 and 3). In fact, a single measurement of hepcidin in a sample using the DRG® hepcidin-25 (Bioactive) ELISA kit (EIA-5258) and the new version of DRG® (EIA-5782) is a single measurement of those kits. Due to the standards provided in, inaccurate results may be obtained.

In addition, the kit produces consistent results when compared to the DRG® comparison kit, despite the anticoagulants used to prepare the plasma (FIGS. 4A and 4B).

This kit is three times wider analytical measurements from zero to 250 ng / ml for the new version of the DRG® and DRG® comparison kits used by the inventors for comparison, 80 ng / ml. It also has a range. This allows a wider range of measurements of samples from diseases known to increase plasma hepsidin levels, such as chronic kidney disease (CKD), dialysis patient samples, and other inflammatory diseases.

The kit has better inter-assay and in-assay coefficient of variation (CV) than that of the DRG® comparison kit.

The reference range reported for this kit shows a clear gender difference between females and males that is not observed with the DRG® kit. Gender differences are a promising feature of the hepcidin reference range for any hepcidin assay that has been shown to be highly effective.

An important difference between the Intrinsic Hepcidin IDx® ELISA kit and the DRG® comparison kit is that the kit is manufactured in an FDA-approved cGMP facility under FDA-compliant design control and quality systems. This kit is suitable for FDA approval after multicenter clinical trials.

An important advantage of the present invention is the sensitive and specific binding of the N-terminus of bioactive hepcidin-25 and other isoforms, which are less bioactive to ferroportin, hepcidin receptors and major iron transporters in humans. A unique high affinity monoclonal antibody, mAb583, used to enable it. Mab583 has an excellent affinity for hepcidin-25 with an estimated Biacore binding constant of 7-8 pM. This mAb583 antibody is conserved as an immortalized hybridoma that is stable and produces a commercial amount of mAb using standard and bioreactor techniques.

On the other hand, little is known about the antibodies used in the comparison kit except those that bind to the C-terminus of hepcidin. The disulfide pair pattern at the C-terminus and in fact at the C-terminus has been shown to have little effect on bioactivity, so testing has shown that binding epitopes that are not specific to the bioactive form of hepcidin. Rely on. Considering the important roles of hepcidin-25 and the N-terminus, the mAb583 antibody used as an important reagent in this kit is significantly improved and excellent. The antibodies used in the DRG® comparison kit are unreliable with respect to the known effects of hepcidin-25 structure and function.

In summary, these examples clearly show that the kit is an improvement over existing commercial kits.

Although preferred embodiments of the invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Here, a number of variations, modifications, and substitutions will be conceived by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the aspects of the invention described herein can be employed in practicing the invention. The appended claims define the scope of the invention, and it is intended by them to include methods and structures within the scope of these claims and their equivalents.

Claims (27)

To diagnose one or more diseases or disorders associated with elevated levels of hepcidin, decreased levels of hepcidin, increased levels of iron, decreased levels of iron, or a combination thereof in biological samples from subjects. It ’s a kit of
(a) 96 microwell strip plates pre-coated with anti-hepcidin antibody;
(b) Hepcidin-25 standard substance;
(c) First hepcidin-25 control;
(d) Second hepcidin-25 control;
(e) Biotinylated hepcidin-25 tracer;
(f) Streptavidin horseradish peroxidase conjugate;
(g) 3,3', 5,5'-Tetramethylbenzidine (TMB) substrate;
(h) Stop solution;
(i) Wash buffer; and
(j) the sample dilution only contains, and
i) When the subject is male, the kit is used to compare the level of hepcidin quantitatively measured with the kit to a reference level averaging 41.5 ng / ml, or
ii) When the subject is female, the kit is used to compare the level of hepcidin quantitatively measured using the kit to a reference level with an average of 21.9 ng / ml.
A kit that features that . The kit according to claim 1, wherein the anti-hepcidin antibody binds to the N-terminal of a mature human hepcidin 25 amino acid peptide. The anti-hepcidin antibody is a heavy chain CDR1 of SEQ ID NO: 8, a heavy chain CDR2 of SEQ ID NO: 9, a heavy chain CDR3 of SEQ ID NO: 10, and a light chain CDR1 of SEQ ID NO: 11. The kit according to claim 1 or 2, comprising 12 light chain CDR2 and SEQ ID NO: 13 light chain CDR3. Any of claims 1 to 3, wherein the anti-hepcidin antibody of (a) contains a heavy chain variable region containing an amino acid sequence of SEQ ID NO: 5 and a light chain variable region containing an amino acid sequence of SEQ ID NO: 7. The kit described in item 1. The kit according to any one of claims 1 to 4, which comprises 8 vials of hepcidin-25 reference material. The kit according to any one of claims 1 to 5, comprising 1 vial of the first hepcidin-25 control. The kit according to any one of claims 1 to 6, comprising 1 vial of a second hepcidin-25 control. The kit according to any one of claims 1 to 7, comprising 1 bottle of biotinylated hepcidin-25 tracer. The biotinylated hepsidin-25 tracer is (i) an oxidatively folded hepsidin peptide; (ii) a hydrophilic spacer consisting of two (2- (2-amino-ethoxy) ethoxy) acetic acid (AEEAc) residues. A hydrophilic spacer in which the hepsidin peptide of (i) is covalently linked to the hydrophilic spacer at the amino end of the peptide; and (iii) covalently linked to the hydrophilic spacer of (ii). The kit according to any one of claims 1 to 8, which comprises biotin. The kit according to claim 9, wherein the oxidatively folded hepcidin peptide has the amino acid sequence described in SEQ ID NO: 1. The kit according to any one of claims 1 to 10, comprising 1 bottle of streptavidin-HRP conjugate. The kit according to any one of claims 1 to 11, which comprises one bottle of TMB substrate. The kit according to any one of claims 1 to 12, which comprises one bottle of stop solution. The kit according to any one of claims 1 to 13, which comprises one bottle of wash buffer. The kit of claim 14, comprising a wash buffer of 20 x solution. The kit of claim 15, wherein the cleaning solution is diluted prior to use in the kit. The kit according to any one of claims 1 to 16, which comprises one bottle of sample diluent. The kit according to any one of claims 1 to 17, further including instructions for use. One or more further including collecting means, kit of claim 1 wherein for the collection of biological samples. 19. The kit of claim 19, wherein one or more collection means comprises a syringe, a needle, a cup, a swab, or a combination thereof. The kit according to claim 19 or 20 , wherein the biological sample comprises a blood sample, a tissue sample or a urine sample. One or more diseases or disorders associated with elevated levels of hemochromatosis, decreased levels of hemochromatosis, increased levels of iron, decreased levels of iron, or a combination thereof, have been associated with African iron overload, alpha salasemia, Alzheimer's disease, anemia, anemia due to cancer, anemia due to chronic diseases, anemia due to inflammation, arteriosclerosis or atherosclerosis, ataxia, iron-related ataxia, atransferrinemia, cancer, celluloplasmin deficiency, chemotherapy Induced anemia, chronic renal disease including end-stage renal disease or chronic renal / renal failure, acute renal injury (AKI), liver cirrhosis, classical hemochromatosis, collagen-induced arthritis (CIA), congenital erythropoiesis Sexual anemia, congestive heart failure, Crohn's disease, Celiac's disease, inflammatory bowel disease (IBD), diabetes, abnormal iron distribution, abnormal iron constancy, abnormal iron metabolism, ferropotin's disease, ferropotin mutant hemochromatosis, folic acid Deficiency, Friedrich ataxia, cord myelopathy, gracile syndrome, bacterial infection, Hallerholden-Spats' disease, hemochromatosis, hemochromatosis caused by mutations in transferrin receptor 2, hemochromatosis, hepatitis, hepatocytes Due to cancer, hereditary hemochromatosis, viral infection, Huntington's disease, hyperferritinemia, hypopigmental microcytic anemia, hypoironemia, insulin resistance, iron deficiency anemia, iron deficiency, iron overload, hepsidin excess Iron deficiency, juvenile hemochromatosis (HFE2), multiple sclerosis, transferrin receptor 2, HFE, hemojuvelin, ferroportin, TMPRSS6 (IRIDA), or mutations in other genes of iron metabolism, neonatal hemochromatosis Hemochromatosis, iron-related neurodegenerative diseases, bone loss, osteoporosis pancreatitis, pantothenate kinase-related neurodegeneration, Parkinson's disease, peragra, heterophagia, porphyrinosis, late-onset cutaneous porphyrinosis, pseudoencephalitis, lung Hemochromatosis, erythrocytosis, rheumatoid arthritis, sepsis, iron blast anemia, systemic erythmatodes, salacemia, intermediate salacemia, infusion iron overload, tumors, vasculitis, vitamin B6 deficiency, vitamin B12 deficiency, Wilson's disease, or a combination thereof, kit of claim 1 wherein. The kit according to any one of claims 1 to 22 , wherein the anti-hepcidin antibody binds to hepcidin with an affinity of ^{10 -11} to 10 ^-14. The kit according to any one of claims 1 to 23 , wherein the anti-hepcidin antibody binds to the N-terminal of hepcidin-25 (SEQ ID NO: 1) with ^{an affinity of 5.16 × 10 -11.} The kit according to any one of claims 1 to 24 for use in a competitive enzyme-bound immunoadsorption assay. The kit according to any one of claims 1 to 25 for use in detecting hepcidin in a blood sample. The kit according to any one of claims 1 to 26 for detecting hepcidin, which comprises an intraassay and interassay coefficient of variation of less than 5%.

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